We have continued our studies of chromatin structure in the neighborhood of expressed genes, making use of the globin gene family in chicken erythrocytes as a model system. We have examined in detail the structure of the nuclease hypersensitive domain in the 5' flanking region of the adult beta globin gene isolated from cells in which that gene is expressed. We have shown that multiple sequence-specific factors are bound in this region, and we have partially purified and characterized some of them. Deletion analysis shows that at least one of these factors is a negative regulatory element that depresses beta globin gene transcription, while another is stimulatory. We have also analyzed the enhancer domain at the 3' side of this gene, which we had discovered earlier. The DNA positive regulatory elements of the enhancer are contained in a region 136 base pairs long, and four discrete sets of cellular factors have been shown by footprint analysis to bind there. Using the primary erythrocyte transfection techniques which we developed, we have shown that the enhancer, in combination with the beta promoter, functions less effectively in 4 day embryonic erythrocytes than in 9 day erythrocytes, strongly suggesting that the enhancer/promoter combination is developmentally specific. We have continued the refinement of the transfection techniques, and determined details of the transfection mechanism. We have also continued physicochemical studies of chromatin structure. We have shown that when an alternating G-C DNA segment contained in a plasmid is converted to the left-handed A form by supercoiling, there is a shift in the placement of nucleosome core particles assembled on the plasmid, in such a way that the Z DNA segment is excluded from the central portion of the nucleosome. The result suggests that this may be a general property of non-B DNA structures.